Coulter Counter Z2 - Users' Server

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Coulter CounterFootprint43 cm W x 45 H cm x 63 depthInstructor and Student Protocol(Preliminary)Link to Beckman Coulter Counter Z series Manual – pdfPrepared by: Bob MorrisonFVCC, Instrumentation SpecialistSep 2008FVCC:BioTech;Morrison 6/22/2010Page 1

Waste canisterCoulter Counter: Basic ControlsRaise/lower mixer in accuvetteClenz canisterMixer; control speedDoor; swing open to accessaccuvette, mixer, and apertureKeypad: Menu displayand control activationPush to raise/loweraccuvette platformFVCC:BioTech;Morrison 6/22/2010Page 2

Coulter Principle : DescriptionIn a Coulter counter, a tube with a small aperture on the wall is immersed into a beaker that contains particles suspended in alow concentration electrolyte. Two electrodes, one inside the aperture tube and one outside the aperture tube but inside thebeaker, are placed and a current path is provided by the electrolyte when an electric field is applied (Figure 1). The impedancebetween the electrodes is then measured. The aperture creates what is called a “sensing zone." Particles in low concentration,suspended in the electrolyte, can be counted by passing them through the aperture. As a particle passes through the aperture,a volume of electrolyte equivalent to the immersed volume of the particle is displaced from the sensing zone. This causes ashort-term change in the impedance across the aperture. This change can be measured as a voltage pulse or a current pulse.The pulse height is proportional to the volume of the sensed particle. If constant particle density is assumed, the pulse height isalso proportional to the particle mass. This technology thus is also called aperture technology.FVCC:BioTech;Morrison 6/22/2010Page 3

Coulter Counter (CC) : Instructor Setup and Testing1.2.3.4.5.6.7.8.The Aperture Tube should be immersed in an accuvette (AV) of CoulterClenz solution (recommended short term storage method). Open the swingdoor for access to the AV. Lower the platform by depressing on the blackbutton on the front and pulling down on the platformRemove (lift) and throw away the Clenz AV solution and prepare another AVwith 10uL of Coulter Isoton solution as a Blank. Gently mix this solution byinversion and place the AV on the platform in the slotted square hole.Depress the black button and raise the platform so that the aperture tube andmetal electrode are immersed in the AV solution. A light should come onwhen the platform is in the fully raised and locked position.Observe the aperture opening in the small display in the upper right corner ofthe device. If necessary, open the swing door and adjust the focus knob.Run the “Fill System” protocol using the Isoton solution.Run the Isoton AV as a blank before student testing to confirm that particlecount is low ( 50).During Student testing, periodically inspect the aperture hole to make sure itis not clogged. Rinse the aperture with DI water between student samples toavoid contamination. (Use a spray bottle and catch spill in extra AV).Run the Isoton blank AV after every 5 samples to ensure that particle countis low.FVCC:BioTech;Morrison 6/22/2010Page 4

Coulter Counter (CC) : Instructor Maintenance and ShutdownNEVER LEAVE the Aperture exposed to air or in a sample with Cells formore than a few hours.1.2.3.4.5.6.Prepare an AV with the Isoton solution of 20ml and place this on theplatform.Run a standard measurement using the Isoton solution.For Maintenance of short term non-use (days) , periodically checkthe level of the solution in the AV and make sure the level is alwaysabove the actual aperture opening, replenish if necessary.Run the Flush System protocol at least once per week.For Maintenance during long-term non-use (weeks), prepare an AVwith the Clenz AV solution and repeat steps 1-3 above.Periodically replenish the AV to keep solution levels covering theaperture at all times.FVCC:BioTech;Morrison 6/22/2010Page 5

Coulter Counter (CC) : Instructor Calibration Setup1.2.3.4.5.6.Each Aperture tube has its own characteristic calibration constant, Kd. In generalonce this is determined and set within the device, it does not have to be redoneunless the tube is improperly stored or contaminated with solutions that leaveresidue on the aperture.To calibrate the aperture tube, use a CC Calibration latex particle solutionselected within 5% to 20% of the aperture opening (found printed on the side ofthe aperture tube). The tube we currently have at FVCC is 100um(Part#9912786). A 10um Calibration Standard Particle solution (Part#6602796)is available as of Aug 2008 (RGM).Prepare the calibration accuvette (AV) by extracting 10mL of the Isoton diluentfrom the storage bottle tap.Add one drop of the Calibration Index solution to this AV, close the cap on theAV, and gently mix by inversion.Open the CC swing door, push the black button, lower the platform, and placethe Calibration solution AV into the platform hole. Return the platform to the topposition and close the glass door.A light should come on inside and a lens display of the aperture opening willappear in the upper right corner of the device. If the aperture opening is notclearly visible, open the swing door and use the focus knob to makeadjustments.FVCC:BioTech;Morrison 6/22/2010 Page 6

Coulter Counter (CC) : Instructor Calibration Execution1.Press the [Setup] membrane button on the control panel. An S1: message willappear called [Enter size Data] showing the aperture diameter, current Kd, andlines for selecting units and setting the upper and lower size expected particlesizes. Use the and v arrows to move to these lines and set new values ifnecessary. Since the NIST standard Calibration solution is non-biological, setthe units as “um”.2.Press [Setup} again and an menu S2: [Enter Analysis Data] appears. If a Kdalready exist in the device for this tube diameter, it will be displayed. Make surethe “Metered Volume” is set to .5 for a 100um aperture tube.3.Press [Setup] to return to the S1 screen, then press [CAL] and the C1 screen willdisplay. Use the arrow keys to set the “Calibrator Size” value to that of theprovided calibrated solution , 10 um. Make sure the “Measure Aperture” line isset to “YES” to force a remeasurement of the aperture paramenter.4.Press [Start] and a C4: Calibration screen will appear with various messagesending in a C3: “Instrument settings” menu.5.Press [Start] and a c6: Calibrate screen appears while the calibration is beingaccomplished. Follow subsequent instructions and/or refer to the device manualSection 3.6.If the Calibration is successful, and C7: Calibration Factor screen will display withthe new calculated Kd value. Press the [Start] button to overwrite and store thisvalue. Press [CAL] if you do not wish to store the new Kd value.Page 7FVCC:BioTech;Morrison 6/22/2010

Coulter Counter (CC) : Student Operation1.2.3.4.5.6.7.8.Prepare Student samples by diluting 1ml of cells in 9ml of Isoton in AV. Otherdilutions may be used, but you need at least 10ml total to trigger apertureoperations. Place a cap on the AV until immediately before running a count.Students may access the Isoton from the CC pump dispenser by removing thecap and unscrewing (CCW) the tap. Raise the pump to the desired level anddepress. Be sure to replace the cap screw when finished.Gently mix the sample 2-3 times by inversion immediately before placing in onthe instrument stand.Depress the SETUP button and verify or set Tupper and Tlower parameters peryour instructors guidance on the S1 screen. Depress the SETUP button again tomove to the S2 screen.On the S2 screen, verify or set the Aperture and Metered Volume and inspect tosee that the Aperture Measure is set to no , and Optimize to yes . Do notchange the Kd value. It has been previously set by the instructor using aCalibration protocol.Depress the Start/Stop button one or more times to START the cell count.On the A4 Screen, record the count data, and depress the Start/Stop button torun at least 3 measurements on a given sample.Rinse the Aperture with DI before releasing the CC for the next student.FVCC:BioTech;Morrison 6/22/2010Page 8

Coulter Counter: Reference InformationInformation on Cell Size, Consumables, andUse of the Coulter for Blood Analysis is presentedIn the following slides. (RGM)FVCC:BioTech;Morrison 6/22/2010Page 9

Coulter CounterInternal Pump andTubing Configuration(taken by RGM 4/7/09 investigating“metering pump failure”FVCC:BioTech;Morrison 6/22/2010Page 10

Part No.Coulter Counter: Reagents and ControlsDescriptionZ Series Starter Kit (Necessary for setup and installation)1 pk.: Z Series Tri-Pak200 ea: Accuvettes2 ea: Sensors1 ea: Calibrator1 ea: COULTER CLENZ 1 ea: Dispenser8320312Z Series Tri-Pak (3 Z Paks) (1 pk.)8320319Z Series Z Pak Conversion Kit (1 ea.))8546719ISOTON II Diluent (20 L)8546929COULTER CLENZ (500 ml)8546930COULTER CLENZ (5 L)8546931COULTER CLENZ (10 L)8320307Diluent Sensor Assembly (for use with Z Series Z Pak)8320308Waste Sensor Assembly (for use with Z Series Z Pak)832030910ml Dispenser (Adjustable Volume Pipette) for use with Z177495Aperture Instrument Concentration Control (1 x 20 ml)Part 8321695832169638348083205926600703Dispersant IA Nonionic (5 x 15 ml)6600704Dispersant IB Nonionic (5 x 15 ml)6600707Dispersant IIIA Cationic (5 x 15 rds Mixed Kit: 1 ea: 2µm, 5µm, 10µm, 20µm, 43µm (5 x 15 ml)L1 Standard, nominal 1µm Latex Particle (NIST Traceable) (1 x 15 ml)L2 Standard, nominal 2µm Latex Particle (NIST Traceable) (1 x 15 ml)L3 Standard, nominal 3µm Latex Particle (NIST Traceable) (1 x 15 ml)L5 Standard, nominal 5µm Latex Particle (NIST Traceable) (1 x 15 ml)L10 Standard, nominal 10µm Latex Particle (NIST Traceable) (1 x 15 ml)L15 Standard, nominal 15µm Latex Particle (NIST Traceable) (1 x 15 ml)L20 Standard, nominal 20µm Latex Particle (NIST Traceable) (1 x 15 ml)L30 Standard, nominal 30µm Latex Particle (NIST Traceable) (1 x 15 ml)L43 Standard, nominal 43µm Latex Particle (NIST Traceable) (1 x 15 ml)L65 Standard, nominal 65µm Latex Particle (NIST Traceable) (1 x 15 ml)L90 Standard, nominal 90µm Latex Particle (NIST Traceable) (1 x 15 ml)Zap-Oglobin IIFVCC:BioTech;Morrison 6/22/201020 µm, Aperture Tube (1 ea)30 µm, Aperture Tube (1 ea)50 µm, Aperture Tube (1 ea)70 µm, Aperture Tube (1 ea)100 µm, Aperture Tube (1 ea)140 µm, Aperture Tube (1 ea)200 µm, Aperture Tube (1 ea)280 µm, Aperture Tube (1 ea)400 µm, Aperture Tube (1 ea)560 µm, Aperture Tube (1 ea)1000 µm, Aperture Tube (1 ea)2000 µm, Aperture Tube (1 ea)50 µm, Aperture Tube, High Resolution (1 ea)70 µm, Aperture Tube, High Resolution (1 ea)100 µm, Aperture Tube, High Resolution (1 ea)140 µm, Aperture Tube, High Resolution (1 ea)200 µm, Aperture Tube, High Resolution (1 ea)Accuvettes (200/pk)Page 11

Cell Size; Mouse FibroblastFVCC:BioTech;Morrison 6/22/2010Page 12

Cell, Bacteria, Virus, Particle Size; ExamplesThickness 200um15um.2um200nmFVCC:BioTech;Morrison 6/22/2010Page 13

Complete Blood Counthttp://www.medicinenet.com/complete blood count/article.htm White blood cell count (WBC). The number of white blood cells in a volume of blood.Normal range varies slightly between laboratories but is generally between 4,300 and10,800 cells per cubic millimeter (cmm). This can also be referred to as the leukocyte countand can be expressed in international units as 4.3 - 10.8 x 109 cells per liter.Automated white cell differential. A machine generated percentage of the different types ofwhite blood cells, usually split into granulocytes, lymphocytes, monocytes, eosinophils,and basophils.Red cell count (RBC). The number of red blood cells in a volume of blood. Normal rangevaries slightly between laboratories but is generally between 4.2 - 5.9 million cells/cmm.This can also be referred to as the erythrocyte count and can be expressed in internationalunits as 4.2 - 5.9 x 1012 cells per liter. Hemoglobin (Hb). The amount of hemoglobin in a volume of blood. Hemoglobin is theprotein molecule within red blood cells that carries oxygen and gives blood its red color.Normal range for hemoglobin is different between the sexes and is approximately 13 - 18grams per deciliter for men and 12 - 16 for women (international units 8.1 - 11.2millimoles/liter for men, 7.4 - 9.9 for women).Hematocrit (Hct). The ratio of the volume of red cells to the volume of whole blood. Normalrange for hematocrit is different between the sexes and is approximately 45 - 52% for menand 37 - 48% for women.FVCC:BioTech;Morrison 6/22/2010Page 14

Complete Blood Count (continued)http://www.medicinenet.com/complete blood count/article.htmMean cell volume (MCV). The average volume of a red cell. This is a calculated valuederived from the hematocrit and red cell count. Normal range is 86 - 98 femtoliters.Mean cell hemoglobin (MCH). The average amount of hemoglobin in the average red cell.This is a calculated value derived from the measurement of hemoglobin and the red cellcount. Normal range is 27 - 32 picograms.Mean cell hemoglobin concentration (MCHC). The average concentration of hemoglobin ina given volume of red cells. This is a calculated volume derived from the hemoglobinmeasurement and the hematocrit. Normal range is 32 - 36%.Red cell distribution width (RDW). A measurement of the variability of red cell size. Highernumbers indicate greater variation in size. Normal range is 11 - 15.Platelet count. The number of platelets in a volume blood. Platelets are not complete cells,but actually fragments of cytoplasm from a cell found in the bone marrow called amegakaryocyte. Platelets play a vital role in blood clotting. Normal range varies slightlybetween laboratories but is in the range of 150,000 - 400,000/ cmm (150 - 400 x 109/liter).FVCC:BioTech;Morrison 6/22/2010Page 15

Link to Beckman Coulter Counter Z series Manual . lines for selecting units and setting the upper and lower size expected particle sizes. Use the and v arrows to move to these lines and set new values if . Follow subsequent instructions and/or refer to the device manual Section 3. 6. If the Calibration is successful, and C7: Calibration .

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